Novel method of selecting immunosuppressant having little thrombocytopenic effect

ABSTRACT

The invention relates to a novel method for selecting an immunosuppressive agent with a less thrombocytopenia effect. According to the invention, a method for selecting an immunosuppressive agent which has a potent immunosuppressive activity but a lower thrombocytopenia effect, said method comprising measuring an IL-2 transcription inhibitory activity in a test cell in to which an IL-2 reporter gene has been introduced in the coexistence of an analyte, while measuring a GATA-1 transcription inhibitory activity in the test cell into which a GATA-1 reporter gene has been introduced in the coexistence of an analyte, and comparing both the transcription inhibitory activities, is provided.

TECHNICAL FIELD

The present invention relates to a novel method for selecting animmunosuppressive agent having the reduced risk of causingthrombocytopenia.

BACKGROUND ART

Major immunosuppressive agents, cyclosporin A (CsA) and tacrolimus(KF506), which have now widely been used in clinical fields in order tosuppress acute rejection after organ transplantation, inhibit theactivity of calcineurin, a Ca²⁺/calmodulin-dependent proteinphosphatase, through binding to respectively specific immunophilins (forexample, cyclophilin for CsA and FKBP12 for FK506). Consequently, it isknown that the intranuclear transfer of NF-AT (nuclear factor ofactivated T-cell) is inhibited by inhibition of dephosphorylation ofNF-AT, resulting in suppression of the transcriptional activity of IL-2gene. From the study of such action mechanism, it has become apparentthat the expression of IL-2 gene at the transcriptional level isinhibited in the activated T-cells to suppress the rejection of thegraft in organ transplantation, and this is very important in obtaininga therapeutic effect in various autoimmune diseases.

In this connection, it has been known that a series of histonedeacetylases (hereinafter referred to as HDAC) that catalyze histonedeacetylation work competitively with histone acetylases in a cellnucleus to control the expression level of various genes throughalteration of the chromatin structure. As the results of so farenergetic screening, a large number of HDAC-inhibitory compounds havebeen provided, which include compounds remarkably inhibiting theproduction of IL-2 (I. Takahashi et al., (1995) The Journal ofAntibiotics 49, 453-457) and have attracted a considerable attention ascandidates of immunosuppressive agents complementing cyclosporin A andtacrolimus. In fact, among thus chosen compounds, some ones showing anexcellent in vivo immunosuppressive effect have been found. For example,as disclosed in WO 00/08048, FR225497 has been found to show anexcellent effect as a therapeutic or preventive agent for organtransplant rejection or autoimmune diseases through the potentimmunosuppressive effect, and in addition, it is suggested to haveusefulness as a therapeutic or preventive agent for many other diseaseswhich are considered to onset due to abnormal expression of genes. Suchdiseases include, for example, inflammatory disorders, diabetesmellitus, diabetic complication, homozygous thalassemia, fibrosis,cirrhosis, acute promyelocytic leukemia (APL), protozoal infection,tumor, and the like. Even though such usefulness has been suggested,however, there is a problem that many of these HDAC-inhibitory compoundswould sometimes have such an adverse reaction as seriousthrombocytopenia when administered to the living body, making itdifficult to use as a practical therapeutic agent.

The cause that many of immunosuppressive HDAC inhibitors readily producesuch a side effect as thrombocytopenia has not yet been elucidatedsufficiently. The present inventors, however, have found in theirassiduous study that many of HDAC inhibitory compounds also inhibit thetranscriptional activity of GATA-1 (also called GATA-1 binding protein,GF-1, NF-E1 or Eryf 1) gene.

GATA-1 is a DNA binding protein which recognizes a (A/T)GATA(A/G)consensus sequence characteristically existing in the transcriptionalregion of hemopoietic gene. This GATA motif sequence has been found in avariety of regulatory regions or promoters such as enhancer regions ofvarious globir genes, locus control regions (LCRs) of β-globin genes,T-cell receptor α-chain, or enhancer regions of δ-chain genes. Inaddition, GATA-1 mRNA has been highly expressed in mature erythrocytes,mast cells or megakaryocytes and slightly expressed in polyfunctionalprecursor cells, or the testicle of young mouse.

In Gata-1 protein, there are 2 sites of C4-type Zn (zinc) finger region.Among them, the Zn finger existing on the N-terminal end has been knownto carry the essential function for mature of erythrocytes andmegakaryocytes through interaction with a transcriptional couplingfactor such as Fog-1 (friend of GATA-1). For example, it has beenreported that, though the human GATA-1 gene is resident on X chromosome,a mutation of V205M or D218G can be recognized at the site correspondingto the Zn finger in some patients suffering from X-chromosomalassociated hereditary dyshemopoietic anemia or thrombocytopenia (K. E.Nichols et al., (2000) Nat. Genet. 24, 266-270; K. Freson et al., (2001)Blood 98, 85-92). Fog-1 per se has 9 Zn fingers, probably through whichit is estimated to play a role in mediating the binding between theGATA-1 linking to a promoter region and the other nuclear factor.

Regarding the promoter of GATA-1 gene itself, at least two promoters,i.e., IE promoter and IT promoter, have so far been found.Correspondingly, there are 2 first exons in this gene. In these exons,it is said that IT promoter specifically acts in the testicle Sertolicells and IE promoter act mainly in hematopoietic cells. However, it hasbeen reported that IT promoter also acts at the step of differentiationof primary erythroid cells (A. M. Vannucchi et al., (1999) Journal ofCellular Physiology 180, 390-401), but it has not yet been elucidatedfully how the 2 promoters are chosen in vivo for action.

In the transcriptional control in hematopoietic cells, the IE promotersequence which is estimated to exist in the range up to around 0.7 kbupstream of the transcription initiation point is considered important.In this region, though there is a sequence coincident with GATA motif orCACC motif, the details of the control sequence present therein arealmost unknown since direct analysis on the human sequence has beenreported very little. However, the transcriptional activity in thisregion alone is very weak, and accordingly it is considered to need theHSI region of about 317 base pairs (highly sensitive region of DNase I)existing at around 3.8 kb to 3.5 kb further upstream, at least inexpression in megakaryocytes (P. Vyas et al., (1999) Development 126,2799-2811; S. Nishimura et al., (2000) Molecular and Cellular Biology20, 713-723). In the HSI region, there is a GATA-E-box motif in whichthe GATA motif sequence is proximate to the E-box motif sequence; thissuggests that the GATA factor such as GATA-1, GATA-2, etc., possiblyforms a complex with a nuclear protein, for example, SCL/ta1-1, E2A(TCF3, transcription factor 3), LMO-2 (LIM only protein 2) or Ldb-1 (LIMdomain binding factor-1) for interaction. In the sequence of the HSIregion, there is a well conserved region between human and mouse.

In a mouse whose GATA-1 gene has been knocked out in a usual way,malformation of primary hematopoietic cells causes lethal damage at thestage of blastogenesis (Y. Fujiwara et al., (1996) Proc. Natl. Acad.Sci. USA 93, 12355-12358). On the other hand, it is possible to preparea knock-out mouse in which the expression of GATA-1 gene ofmegakaryocyte line is selectively knocked out; in such a mouse, it hasbeen found that the number of platelet is sharply reduced and the normalmaturation of megakaryocytic cells is not recognized (R. A. Shivdasaniet al., (1997) The EMBO Journal 16, 3965-3973).

DISCLOSURE OF INVENTION

To date, a large number of HDAC inhibitory compounds exhibiting anexcellent immunosuppressive effect when administered to the living body,are known, but these compounds are not necessarily satisfactory becausethey have serious thrombocytopenia at the same time, making it difficultto use clinically. Thus, it has strongly been desired to provide a goodmethod for in vitro screening a compound from these candidates which hasno thrombocytopenia effect. The purpose of the invention is to solvesuch a problem.

From the studies to date, it has been found that the GATA-1 gene productplays an important role in differentiation and maturation oferythrocytes and megakaryocytic cells (X. Tang et al., (2001): CMLS,Cell. Mol. Life Sci. 58, 2008-2017). In addition to GATA-1, however,many other factors have been suggested to be involved in differentiationand maturation of megakaryocytic cells; and it was really unclearwhether the thrombocytopenia effect often recognized in many of HDACinhibitors is through inhibition of the function of GATA-1 itself.According to the present inventor's study, it was first elucidated, as aresult of comparative study on the effect of various HDAC inhibitors,that the thrombocytopenia effect caused by administration of these drugsis based on suppression of the transcription of GATA-1 gene. In order tosearch the cause of thrombocytopenia effect observed in many of HDACinhibitors, the present inventors, using GeneChip® (Affymetrix),screened and grouped a human-originated gene of which the transcriptionwas inhibited by an HDAC inhibitor in the same pattern as GATA-1 fromthe genes expressing in megakaryocytic cells, and searched atranscriptional factor common to these gene groups. And, from 45 genesof which the transcription was inhibited by an HDAC inhibitor in thesame pattern as GATA-1 gene, the inventors obtained 10 genes ascandidates for a transcriptional factor expected to have the functioninvolved in differentiation of blood corpuscles or as candidatesexpected to be megakaryocytic marker genes. Then, the inventors examinedin details whether or not a binding sequence of a transcriptional factorcommonly present in the transcriptional control region of the respectivegenes and that of the GATA-1 gene exists, and they found that there is aresponsive sequence recognized by STAT3, C/EBPβ and HSF1 almost commonlyin addition to the responsive sequence recognized by GATA-1 itself (GATAsequence). The inventors then constructed an expression system for areporter gene which is regulated by the responsive sequence recognizedby other respective transcriptional factors than the above-mentionedGATA-1, and examined the effect with HDAC inhibitors. In any cases, itwas found that the transcription of reporter gene was not inhibited byHDAC inhibitors.

Further, the present inventors constructed artificially a promoter inwhich a responsive sequence recognized by GATA-1 itself was lost, byintroducing a mutation into the GATA sequence present in the GATA-1 genepromoter. This was integrated in an expression system for a reportergene and transformed into cells to examine. As a result, it was foundthat transcription of the reporter gene was not inhibited by at least 3members of HDAC inhibitors. These results indicate that the inhibitionof GATA-1 gene expression by HDAC inhibitors is caused by inhibition ofthe function of transcriptional activation by transcriptional factorsthrough a GATA sequence in the GATA-1 gene promoter. That is, theseresults strongly suggest a possibility that the inhibition by an HDACinhibitor of the transcriptional activation function induced by a GATA-1factor might generate an adverse effect, thrombocytopenia, since theGATA-1 factor per se exhibits the transcriptional activation functionthrough the GATA sequence.

In this connection, the present inventors have eagerly studied aneffective dose of a large number of HDAC inhibitors showing animmunosuppressive activity on a rat's heart transplantation model, andthey have realized that the efficacy as an immunosuppressive agent inthe rat's heart transplantation model is deeply associated withinhibition of the IL-2 gene expression. In order to confirm this fact, areporter assay system was constructed using a transcriptional controlregion of IL-2 gene. A DNA construct containing the reporter gene wasintroduced into an activated cell strain derived from T-cell, and achange of the reporter activity was measured and compared with additionof a variety of compounds to be tested. As a result, it was found thatthe efficacy as an immunosuppressive agent in the rat's hearttransplantation model correlated well with the inhibition of IL-2transcription.

Further, the present inventors have realized that the serious sideeffect of a large number of HDAC inhibitors, i.e., thrombocytopeniaeffect is deeply associated with the inhibition of expression of GATA-1gene. In order to confirm this fact, a reporter assay system wasconstructed using a transcriptional control region of GATA-1 gene. A DNAconstruct containing these reporter genes was introduced into a cellstrain derived from a megakaryocytic cells, and a change of the reporteractivity was measured and compared with addition of a variety ofcompounds (analytes) to be tested. As a result, it was found that therewas a tendency that a compound having a stronger platelet inhibitoryactivity strongly inhibited the transcription of GATA-1 gene.

In this connection, the present inventors have found a method forselecting an immunosuppressive agent with a less thrombocytopeniaeffect, the method comprising making an analyte coexist with a test cellinto which a GATA-1 reporter gene has been introduced, and measuring thetranscription inhibitory activity of the GATA-1 in the test cell.

Further, the inventors invented a method for rapidly selecting acompound with a less thrombocytopenia effect as a side effect and withan immunosuppressive activity, the method comprising selecting acompound having a strong inhibitory activity for IL-2 transcription anda weak inhibitory activity for GATA-1 transcription by simultaneouslyusing a reporter assay system utilizing the above 2 species of cells.

The invention relates to a method for selecting an immunosuppressiveagent with a less thrombocytopenia effect, the method comprisingmeasuring an IL-2 transcription inhibitory activity in a test cell intowhich an IL-2 reporter gene has been introduced in the coexistence of ananalyte, while measuring a GATA-1 transcription inhibitory activity inthe test cell into which a GATA-1 reporter gene has been introduced inthe coexistence of an analyte, and comparing both the transcriptioninhibitory activities.

Further, the invention relates to a method for selecting animmunosuppressive agent with a less thrombocytopenia effect, the methodcomprising measuring the amount of expressed IL-2 protein, measuring theamount of expressed GATA-1 protein, and comparing both the amounts ofexpression.

Further, the invention relates to a kit for selecting animmunosuppressive agent with a less thrombocytopenia effect, the kitcomprising a DNA construct containing an IL-2 reporter gene, a DNAconstruct containing a GATA-1 reporter gene, a test cell of T-cell line,and a test cell of megakaryocytic cell line.

Further, the invention relates to a therapeutic agent for treatment ofinflammatory disorders, diabetes mellitus, diabetic complications,homozygous thalassemia, fibrosis, cirrhosis, acute promyelocyticleukemia (APL), protozoal infections, organ transplant rejection,autoimmune diseases, and tumors, the agent comprising as an activeingredient a compound selected by measuring the (IL-2 IC50) value as anIL-2 transcription inhibitory activity, measuring the (GATA-1 IC50)value as a GATA-1 transcription inhibitory activity, comparing both thevalues, and selecting a compound having the (GATA-1 IC50)/(IL-2 IC50)value of 5 or more.

That is, the present invention relates to the following ones.

<1> A method for selecting an immunosuppressive agent with a lessthrombocytopenia effect, the method comprising the following items (1)and (2):

(1) selecting compounds having an immunosuppressive activity; and

(2) selecting a compound having a weak GATA-1 transcription inhibitoryactivity from the compounds selected in (1).

<2> A method for selecting an immunosuppressive agent with a lessthrombocytopenia effect, the method comprising the following items (1)to (3):

(1) measuring an immunosuppressive activity of an analyte;

(2) measuring a GATA-1 transcription inhibitory activity of the analyte;and

(3) comparing the immunosuppressive activity determined in (1) with theGATA-1 transcription inhibitory activity determined in (2) to select animmunosuppressive agent with a less thrombocytopenia effect.

<3> A method for selecting an immunosuppressive agent with a lessthrombocytopenia effect, the method comprising the following items (1)to (3):

(1) measuring an IL-2 transcription inhibitory activity in a test cellin the coexistence of the test cell and an analyte;

(2) measuring an GATA-1 transcription inhibitory activity in a test cellin the coexistence of the test cell and an analyte; and

(3) comparing the IL-2 transcription inhibitory activity determined in(1) with the GATA-1 transcription inhibitory activity determined in (2)to select an immunosuppressive agent with a less thrombocytopeniaeffect.

<4> A method for selecting an immunosuppressive agent with a lessthrombocytopenia effect, the method comprising the following items (1)to (3):

(1) measuring an IL-2 transcription inhibitory activity in a test cellinto which an IL-2 reporter gene has been introduced in the coexistenceof the test cell and an analyte;

(2) measuring a GATA-1 transcription inhibitory activity in a test cellinto which a GATA-1 reporter gene has been introduced in the coexistenceof the test cell and an analyte; and

(3) comparing the IL-2 transcription inhibitory activity determined in(1) with the GATA-1 transcription inhibitory activity determined in (2)to select an immunosuppressive agent with a less thrombocytopeniaeffect.

<5> A method as described in <4> for selecting an immunosuppressiveagent with a less thrombocytopenia effect, comprising measuring a (IL-2IC50) value as an IL-2 transcription inhibitory activity, measuring a(GATA-1 IC50) value as a GATA-1 transcription inhibitory activity, andcomparing both the values.

<6> A method as described in <5> for selecting an immunosuppressiveagent with a less thrombocytopenia effect, comprising selecting acompound having the (GATA-1 IC50) (Il-2 IC50) value of 5 or more.

<7> A method as described in any of <4> to <6>, wherein the GATA-1reporter gene comprises the transcriptional control region of humanGATA-1 gene and a reporter gene.

<8> A method as described in any of <4> to <7>, wherein the GATA-1reporter gene comprises a sequence of the region from −3769 to −3133upstream of the transcription initiation point and sequence of theregion from −789 to +30 proximal to the transcription initiation pointof human GATA-1 gene.

<9> A method as described in any of <4> to <8>, wherein the IL-2reporter gene comprises the transcriptional control region of IL-2 geneand a reporter gene.

<10> A method as described in any of <4> to <9>, wherein the IL-2reporter gene comprises a sequence of the region from −378 to +54proximal to the transcription initiation point of human IL-2 gene.

<11> A method as described in any of <4> to <10>, wherein the test cellinto which a GATA-1 reporter gene is introduced is a humanmegakaryocytic cell strain.

<12> A method as described in any of <4> to <11>, wherein the test cellinto which an IL-2 reporter gene is introduced is a human T cell-derivedcell strain stimulated by phorbol 12-myristate 13-acetate, ionomycin andanti-CD28 antibody, and the test cell into which a GATA-1 reporter geneis introduced is a human megakaryocytic cell strain.

<13> A method as described in claim 12, wherein the human T cell-derivedcell strain is a Jurkat cell.

<14> A method as described in <11>, wherein the human megakaryocyticcell strain is a HEL cell.

<15> A method as described in <12>, wherein the human megakaryocyticcell strain is a HEL cell.

<16> A method as described in any of <4> to <15>, wherein the reportergene is a firefly luciferase gene.

<17> A method for selection as described in any of <1> to <16>, whereinthe analyte is an HDAC inhibitor.

<18> A method for selecting an immunosuppressive agent with a lessthrombocytopenia effect, comprising measuring the amount of expressionof GATA-1 protein.

<19> A method for selecting an immunosuppressive agent with a lessthrombocytopenia effect, the method comprising the following items (1)to (3):

(1) measuring the amount of expression of IL-2 protein;

(2) measuring the amount of expression of GATA-1 protein; and

(3) comparing both the amounts of expression to select animmunosuppressive agent with a less thrombocytopenia effect.

<20> A kit for measurement in selecting an immunosuppressive agent witha less thrombocytopenia effect, comprising the following items (1) and(2):

(1) a DNA construct containing a GATA-1 reporter gene; and

(2) a test cell of megakaryocytic cell line.

<21> A kit for measurement in selecting an immunosuppressive agent witha less thrombocytopenia effect, comprising the following items (1) to(4):

(1) a DNA construct containing an IL-2 reporter gene;

(2) a DNA construct containing a GATA-1 reporter gene;

(3) a test cell of T-cell line; and

(4) a test cell of megakaryocytic cell line.

<22> An HDAC inhibitor with a less thrombocytopenia effect, theinhibitor being selected by a method as described in <17>.

<23> An immunosuppressive agent with a less thrombocytopenia effect, theagent being selected by a method for selection as described in any of<1> to <16>.

<24> An immunosuppressive agent for treatment of inflammatory disorders,diabetes mellitus, diabetic complications, homozygous thalassemia,fibrosis, cirrhosis, acute promyelocytic leukemia, protozoal infections,organ transplant rejection, autoimmune diseases, and tumors, the agentcomprising as an active ingredient an HDAC inhibitor as described in<22>.

<25> A therapeutic agent for treatment of inflammatory disorders,diabetes mellitus, diabetic complications, homozygous thalassemia,fibrosis, cirrhosis, acute promyelocytic leukemia, protozoal infections,organ transplant rejection, autoimmune diseases, and tumors, the agentcomprising as an active ingredient an immunosuppressive agent asdescribed in <23>.

<26> A therapeutic method for treatment of inflammatory disorders,diabetes mellitus, diabetic complications, homozygous thalassemia,fibrosis, cirrhosis, acute promyelocytic leukemia, protozoal infections,organ transplant rejection, autoimmune diseases, and tumors, the methodcomprising administering an immunosuppressive agent with a lessthrombocytopenia effect containing as an active ingredient an HDACinhibitor as described in <22>.

<27> A therapeutic method for treatment of inflammatory disorders,diabetes mellitus, diabetic complications, homozygous thalassemia,fibrosis, cirrhosis, acute promyelocytic leukemia, protozoal infections,organ transplant rejection, autoimmune diseases, and tumors, the methodcomprising administering an immunosuppressive agent with a lessthrombocytopenia effect containing as an active ingredient animmunosuppressive agent as described in <23>.

<28> Use of an HDAC inhibitor as described in <22> in manufacture of atherapeutic agent for treatment of inflammatory disorders, diabetesmellitus, diabetic complications, homozygous thalassemia, fibrosis,cirrhosis, acute promyelocytic leukemia, protozoal infections, organtransplant rejection, autoimmune diseases, and tumors.

<29> Use of an immunosuppressive agent as described in <23> inmanufacture of a therapeutic agent for treatment of inflammatorydisorders, diabetes mellitus, diabetic complications, homozygousthalassemia, fibrosis, cirrhosis, acute promyelocytic leukemia,protozoal infections, organ transplant rejection, autoimmune diseases,and tumors.

The invention will be explained in detail as follows.

The invention relates to a method for selecting an immunosuppressiveagent with a less thrombocytopenia effect, the method comprisingmeasuring an IL-2 transcription inhibitory activity in a test cell intowhich an IL-2 reporter gene has been introduced in the coexistence of ananalyte, while measuring a GATA-1 transcription inhibitory activity inthe test cell into which a GATA-1 reporter gene has been introduced inthe coexistence of an analyte, and comparing both the transcriptioninhibitory activities.

The term “IL-2 reporter gene” refers to a DNA construct in which atranscriptional control region of IL-2 gene is ligated artificially to areporter gene. As the transcriptional control region of IL-2 gene, forexample, a sequence comprising a proximal region of the transcriptioninitiation point of IL-2 gene (GenBank Accession Number: X00695, Locuscode: HSIL05) and its upstream region may be used. As the scope of thetranscriptional control region necessary for the purpose of theinvention, it is desired to not only hold a transcriptional activity butalso substantially reflect the transcriptional control mode of a naturalIL-2 gene in an active-type T cell. In fact, it has been reported that,in a human IL-2 gene, if a region of about 275 base pairs is present atthe position upstream of the transcription initiation point, then itwould function as a promoter reflecting substantially thetranscriptional control mode of a natural IL-2 gene in a cell strainJurkat (JCRB0062, Japanese Collection of Research Bioresources) derivedfrom human T-lymphoblast (D. B. Durand et al., (1987) J. Exp. Med. 165,395-407). From the studies to date, it has been found that the bindingsite of proteins such as NF-AT, OCT-1 (octamer-binding transcriptionfactor-1), NF-κB, AP-1, CD28RC (CD28 responsive element bindingcomplex), ZEB (TCF8, NIL-2-A zinc finger protein), and the like, isplaced in this region. Therefore, if the region contains an essentialregion for such protein binding, any length of sequence could be used asthe region for use in construction of an IL-2 reporter gene; forexample, the region of 434 base pairs as described in Sequence Listing 9(when the major transcription initiation point is +1, the regioncorresponds to from −378 to +54) may be used.

In order to establish a screening system for immunosuppressive agentseffective to human, it is desired to use a transcriptional controlregion of an IL-2 gene of human origin. For the screening of compoundseffective in a model animal such as mouse or rat, the transcriptionalcontrol region of IL-2 gene derived from each animal may be used, andsuch a DNA construct can easily be constructed by a person skilled inthe usual experimental technique.

In the above-mentioned invention, the term “GATA-1 reporter gene” refersto a DNA construct in which a transcriptional control region, of GATA-1gene is ligated artificially to a reporter gene. As the transcriptionalcontrol region of GATA-1 gene, for example, a DNA fragment comprisingboth an IE promoter region of GATA-1 gene (GenBank Accession Number:AF196971) and an HSI region (highly sensitive region to DNase I) atabout 3.5 kb separated from its upstream region may be used. As thescope of the transcriptional control region necessary for the purpose ofthe invention, it is desired to not only merely hold a transcriptionalactivity but also substantially reflect the transcriptional control modeof a natural GATA-1 gene in megakaryocytic cells.

As mentioned above, it is known that as a promoter of GATA-1 gene thereare at least 2 kinds of promoters, i.e., IE promoter and IT promoter.Among them, IE promoter is known to have a close relation with thetranscriptional control in the megakaryocytic cells in relation with athrombocytopenia effect by an HDAC inhibitor; thus, the function of IEpromoter is estimated to be important, and its control region isutilized in this invention.

For the transcriptional control by IE promoter, a sequence presentwithin 0.7 kb just upstream of the transcription initiation point andthe further upstream HSI region (highly sensitive region to DNase I)both are important. There are 2 CACC sites at 5′ end of the HSI region,and at the position separated about 50 base pairs from the HSI region,there are a GATA binding site and an E-box motif side by side andseparated about 10 base pairs from each other. It has been known thatwhen a mutation is introduced so as to destroy the GATA binding site inthis situation, no expression specific to erythrocytes or megakaryocytescan be observed (P. Vyas et al., (1999) Development 126, 2799-2811). Inaddition, at the 3′ end of GATA-E-box motif, a characteristic palindromesequence CTGTGGCCACAG sequence and a region rich in GC are present. Inthe region rich in GC, a GGAA sequence seen in the ETS binding site of atranscriptional factor is present. In the GATA-1 HSI region containingthese sequences, even though the 5-side CACC site is deleted, theexpression specific to erythrocytes or megakaryocytes can be observed,but when the GATE-E-box motif is deleted, no expression is observed. Atthe 3′ end, it has been reported that those containing at most about 250base pairs at downstream of E-box function as an enhancer. Therefore, ifall of these essential sequence sites are involved, any length of thesequence might be accepted practically; for example, it is possible touse a sequence in which a region containing 819 base pairs proximal tothe transcription initiation point (from −789 to +30; the transcriptioninitiation point is fixed as +1) by IE promoter of human GATA-1 gene isligated artificially to a region containing 637 base pairs (from −3769to −3133) as an HIS region at upstream of the transcription initiationpoint.

Regarding an essential sequence site such as GATE-E box motif, it ispossible to prepare an artificial DNA construct in which a multiple ofthe essential sequences are linked together in tandem by a conventionalrecombinant DNA experimental technique. When such an artificialconstruct is used in place of a natural sequence, the transcriptionalinduction activity in the transcriptional control region can sometimesbe increased.

In order to confirm a relationship between the GATE-1 transcriptioninhibitory activity of various HDAC inhibitors and the thrombocytopeniaeffect in an animal model such as mouse or rat, a similar DNA constructmay be prepared using the transcriptional control region of GATA-1 geneof animal origin in place of the above-mentioned human sequence; such aDNA construct can easily be constructed by a person skilled in the usualexperimental technique.

The term “reporter gene” as used in the invention refers to a gene whichcomprises a structural gene region coding for a specific protein as amarker of the gene expression and a downstream 3′-noncoding region andin which the transcriptional control regions originally present at theupstream of the structural genes are deleted all. As for the structuralgene region which codes for a specific protein serving as a marker ofthe gene expression, any type of ones may be used as far as the geneexpression from the transcriptional control region contained in anextraneous gene fragment can easily be measured from the function of amarker protein corded by the reporter gene when it is artificiallyligated to the extraneous gene fragment and introduced into a cell. Morepreferably, it is to be desirable that the protein as a marker can existstably in mammal cells and no intrinsic protein having a similaractivity exists in the cell or it can easily be distinguished from themarker even though it exists in the cell. More preferably, it is to bedesirable that mRNA coding for a marker protein exists stably in cells.It is also desirable that in measurement of the activity, the substratefor the marker protein is not required to be added additionally oreasily incorporated sufficiently in the cells even if addition isrequired. Further, the measurement system composed of these elements isdesired to provide linearity in a wide range and high sensitivity.Luciferase gene of firefly origin, luciferase gene of Renilla origin,alkaline phosphatase, β-galactosidase gene, green fluorescent protein(GFP) gene, enhanced green fluorescent protein (EGFP) gene,β-glucuronidase gene, chloramphenicol acetyltransferase (CAT) gene,horse radish peroxidase (HRP) gene, and the like may be used. Morepreferably, luciferase gene of firefly origin may be used. As forluciferase gene of firefly origin, an improved type of luciferase gene(luc+) may be used to increase the detection sensitivity of ameasurement system. For the non-coding region downstream of thestructural gene, for example, a sequence derived from SV40 virus genomelate gene may be used.

The above-mentioned IL-2 reporter gene and GATA-1 reporter gene arerespectively ligated to a cloning vector and multiplied in Escherichiacoli as a host. As a cloning vector used in this operation, a vectorhaving the origin of replication and selective marker amplifiable inEscherichia coli can be used without any limitation; for example,pGL3-Basic (Promega Corporation) coding for an ampicillin resistancemarker and an improved lucifurase structural gene (luc+) may be used toeasily prepare the above-mentioned DNA construct. Thereafter, theamplified vector is introduced transiently into an animal cell tomeasure the reporter activity.

The DNA construct containing a IL-2 reporter gene and GATA-1 reportergene may be introduced by means of transformation utilizing any one of ausual calcium phosphate method, liposome method, lipofectin method, andelectroporation method, without any limitation. More preferably,electroporation may be used.

As for the “test cell” into which an IL-2 reporter gene is introduced asa system for selecting immunosuppressive compounds, Jurkat cells(JCRB0062, Japanese Collection of Research Bioresources) may be used asa host cell, though other cell lines may be used as far as they arethose derived from T cells. From the studies of the action mechanism ofimmunosuppressive agents such as cyclosporin A, tacrolimus, and thelike, it has been known that the inhibition of the IL-2 expression inactivated T-cells correlates well with the suppressive effect of thesedrugs for acute rejection after organ transplantation. Therefore, it isnecessary to use as a test cell a cell substantially reflecting thestate of human T cell during activation but not resting phase, inestablishing a system for evaluating an immunosuppressive effect of ananalyte using an IL-2 reporter gene. In T cells, 2 signals are known,i.e., the first signal which mediates a TCR/CD3 complex recognizing aMHC complex representing CD4/CD8 and an antigenic peptide on the targetcell, and the second signal which mediates CD28 recognizing CD80 or CD86on the target cell. In order to maintain the activation of T cells, itis necessary to give stimulation so as to generate both of the above 2signals at the same time, or give stimulation bypassing them. Thenon-specific activation of T cells caused by the first signal is knownto be accompanied by phosphorylation of a group of proteins or increaseof an intracellular calcium concentration; this effect empirically canbe substituted by addition of a phorbol ester such as 12-myristate13-acetate, or ionomycin. In addition, anti-CD28 antibody may be used asstimulation to the above second signal. The activated test cells used inthe invention, accordingly, can be obtained by simultaneous stimulationof the above-mentioned host cells with phorbol 12 myristate 13 acetate,ionomycin or anti-CD28 antibody.

Similarly, as a “test cell” into which a GATA-1 reporter gene isintroduced in selecting a compound with a less thrombocytopenia effect,it is necessary to use as a host cell a cell strain substantiallyreflecting the state of the cell of megakaryocytic cell line. As thecell line of megakaryocytes, for example, HEL cell (JCRB0062, JapaneseCollection of Research Bioresources) of human origin can be used withoutparticular limitation.

The “analyte” as used in the invention means a candidate substance to bemeasured in the invention, including, but not limited to, low molecularorganic compounds, low molecular inorganic compounds, high molecularcompounds including proteins and nucleic acids, sugars, and all othercompounds, as well as their liquid mixtures, natural products, syntheticproducts, and extracts from animals or fungi, algae, or microorganisms.

In the invention as mentioned above, “the coexistence of the test celland an analyte” may be achieved by adding an analyte to a culture mediumbefore or after introduction of an IL-2 reporter gene or GATA-1 reportergene into a test cell by electroporation. More preferably, about 1 to 24hours after introduction of an IL-2 reporter gene or GATA-1 reportergene, more preferably about 3 to 12 hours after the introduction, ananalyte may be added to the culture medium, and cultured for anadditional 1-24 hours, more preferably for 8-12 hours.

In the invention as mentioned above, “IL-2 transcription inhibitoryactivity” can be determined in the above-mentioned test cell into whichan IL-2 reporter gene has been introduced, by comparing the activity ofthe reporter gene product in the coexistence of an analyte with thestandard activity of the reporter gene product determined in the absenceof an analyte. When the reporter gene product is a sufficiently stableprotein, the activity of this gene product is expected to be roughlyproportional to the transcriptional activity of the IL-2 gene.

Similarly, in the invention as mentioned above, “GATA-1 transcriptioninhibitory activity” can be determined in the above-mentioned test cellinto which a GATA-1 reporter gene has been introduced, by comparing theactivity of the reporter gene product in the coexistence of an analytewith the standard activity of the reporter gene product determined inthe absence of an analyte.

According to the method as mentioned above, a test cell into which anIL-2 reporter gene has been introduced (e.g., Jarkat cell) and a testcell into which a GATA-1 reporter gene has been introduced (e.g., HELcell) can be constructed, respectively. These cells respectively areallowed to coexist with a variety of analytes, and the potency of IL-2transcription inhibitory activity and of GATA-1 transcription inhibitoryactivity can be detected and measured using expression of the respectivereporter genes as indicator. In order to select an immunosuppressiveagent with a less thrombocytopenia effect, a substance whichspecifically inhibits expression of IL-2 but does not inhibit expressionof GATA-1 gene in a test cell system may be selected.

Practically, in screening an effective compound clinically, it isappropriate to evaluate the thrombocytopenia effect of each analyte inbalance with the potency of an immunosuppressive effect; that is, thestandard of evaluation is determined by examining how degree ofthrombocytopenia effect is attained at an effective dose at which acertain level of immunosuppressive effect is recognized, when theanalyte is administered to the living body. Practically, this can becalculated as follows. That is, using the above-mentioned 2 expressionsystems for the reporter genes, the amount of expression of luciferaseis determined. The term “IL-2 IC50” as used in the invention indicatesthe concentration of the added analyte at which the expression of theIL-2 reporter gene is inhibited by 50% when the amount of expression ofIL-2 reporter gene is regarded as 100% where no analyte is added at all.Thus, IC50 can easily be calculated from a drug dose-response curve.This value is expected to correlate with an effective dose at which adrug shows a certain immunosuppressive effect in vivo, that is, aminimum effective dose at which the drug shows an immunosuppressiveeffect. Similarly, the term “GATA-1 IC50” as used in the inventionindicates the concentration of the added analyte at which the expressionof the GATA-1 reporter gene is inhibited by 50% when the amount ofexpression of GATA-1 reporter gene is regarded as 100% where no analyteis added at all. Thus, GATA-1 IC50 can easily be calculated from a drugdose-response curve. Thus calculated GATA-1 IC50 value is divided by theIL-2 IC50 value (i.e., (GATA-1 IC50)/(IL-2 IC50)); the resulting (GATA-1IC50)/(IL-2 IC50) value is expected to correlate with the degree ofthrombocytopenia effect caused by an analyte at a dose at which theanalyte can show a certain degree of immunosuppressive effect whenadministered to a living body. That is, this (GATA-1 IC50)/(IL-2 IC50)value, for example, is expected to correlate with the potency ofthrombocytopenia effect at the minimum effective dose of an analyte atwhich dose an immunosuppressive effect is recognized when administeredto a living body. Practically, the (GATA-1 IC50)/(IL-2 IC50) value ofeach analyte obtained as mentioned above based on the reporter assaydata, has been confirmed to well correlate with the rate of plateletdecrease at a minimum dose at which the analyte administered to a rat'sheart transplant model shows an immunosuppressive effect: In addition,it has been found that, for example, in a compound of which the (GATA-1IC50)/(IL-2 IC50) value is 5 or higher, the rate of platelet decrease issuppressed to 30% or lower at a minimum dose at which animmunosuppressive effect is shown in a rat's heart transplant model.

As mentioned above, it is desirous to determine the “GATA-1transcription inhibitory activity” and “IL-2 transcription inhibitoryactivity” using the activity of the reporter gene product as anindicator, by introducing an artificially constructed GATA-1 reportergene or IL-2 reporter gene into a cell. Alternatively, it is alsopossible to measure the transcriptional activity by determining GATA-1mRNA or IL-2 mRNA by means of RT-PCR or DNA microarray on the cell intowhich the GATA-1 gene or IL-2 gene containing all of the above-mentionedtranscriptional control region per se has been introduced. Depending onsome cell lines, it is also possible to directly monitor the expressionof native GATA-1 gene or IL-2 gene endogenous in cells by means ofRT-PCR or DNA microarray. Such an assay system can easily be establishedingeniously by a person skillful in an experimental technique.

In addition, the invention relates to a method for selecting animmunosuppressive agent with less thrombocytopenia effect, the methodcomprising measuring the amount of expression of IL-2 protein, measuringthe amount of expression of GATA-1 protein, and comparing both theamounts of expression.

In the invention as mentioned above, the “amount of expression of IL-2protein” can be determined by treating an extract of the above-mentionedtest cells containing no IL-2 reporter gene with an analyte, andmeasuring the amount of expression of IL-2 protein with an anti-IL-2antibody and a labeled secondary antibody. As such a technique, aradioactive isotopic immunoassay (RIA), ELISA (E. Engvall et al.,(1980), Methods in Enzymol., 70, 419-439), fluorescence antibodytechnique, plaque technique, spot method, aggregation method,Ouchterlony, and other various methods used in conventionalimmunochemical measurement techniques (“Hybridoma technique andmonoclonal antibodies”, published by R&D Planning, 30-53, March 5,(1982)) can be utilized. These techniques can easily be conducted by aperson skillful in the art. Though the above-mentioned techniques canproperly be chosen from many points of view, ELISA is preferablyemployed in view of sensitivity and convenience.

The labeling substance used in labeling the above secondary antibodyincludes enzymes such as horse radish peroxidase, alkalinephosphatase,etc., fluorescent substances such as fluorescein isocyanate, rhodamine,etc., radioactive substances such as ³²P, ¹²⁵I, etc., andchemiluminescences.

In the same technique, the “amount of expression of GATA-1 protein” maybe determined by using an anti-GATA-1 antibody and a labeled secondaryantibody.

In addition, the invention relates to a kit for measurement in selectingan immunosuppressive agent with a less thrombocytopenia effect,comprising a DNA construct containing an IL-2 reporter gene; a DNAconstruct containing a GATA-1 reporter gene; a test cell of T-cell line;and a test cell of megakaryocytic cell line.

In the invention as mentioned above, the “kit for measurement inselecting an immunosuppressive agent with a less thrombocytopeniaeffect” may be used in measurement by detecting the potency of the IL-2transcription inhibitory activity and of the GATA-1 transcriptioninhibitory activity in a test cell in the coexistence of the test cellinto which an IL-2 reporter gene or GATA-1 gene has been introduced andan analyte, wherein the expression of IL-2 reporter gene or GATA-1reporter gene is used as an indicator of the expression. Specifically,the kit comprises the following items (1) to (4):

(1) a DNA construct containing an IL-2 reporter gene;

(2) a DNA construct containing a GATA-1 reporter gene;

(3) a test cell of T-cell line, more preferably, Jurkat cell; and

(4) a test cell of megakaryocytic cell line, more preferably, HEL cell.

In the invention as mentioned above, the “DNA construct” refers to DNAcontaining the above-described IL-2 reporter gene or GATA-1 reportergene, which may be or may not be incorporated in a cloning vector andmay be cyclic or acyclic.

In addition, the invention relates to a therapeutic method andtherapeutic agent for treatment of inflammatory disorders, diabetesmellitus, diabetic complications, homozygous thalassemia, fibrosis,cirrhosis, acutepromyelocytic leukemia, protozoal infections, organtransplant rejection, autoimmune diseases, and tumors, comprising as anactive ingredient a compounds which is obtained by measuring the (IL-2IC50) value as an IL-2 transcription inhibitory activity, measuring the(GATA-1 IC50) value as a GATA-1 transcription inhibitory activity, andcomparing both of the values to select a compound of which the (GATA-1IC50)/(IL-2 IC50) value is 5 or higher.

According to the above-mentioned method for evaluation, the (IL-2 IC50)value is measured as an IL-2 transcription inhibitory activity, the(GATA-1 IC50) value is measured as a GATA-1 transcription inhibitoryactivity, and both of the values are compared to select a compound ofwhich the (GATA-1 IC50)/(IL-2 IC50) value is 5 or higher; thus resultingcompound is useful as an immunosuppressive agent with lessthrombocytopenia effect in treatment or prevention of organ transplantrejection or autoimmune diseases. In addition, it is also useful intreatment or prevention of the following diseases possibly caused byabnormal gene expression: inflammatory disorders, diabetes mellitus,diabetic complications, homozygous thalassemia, fibrosis, cirrhosis,acute promyelocytic leukemia, protozoal infections, and tumors.

When these compounds are used as drugs in the invention, they can beused per se as drugs, or alternatively they may be formulated intopharmaceutical preparations according to a conventional pharmaceuticaltechnology. For example, they may be properly combined with (a)pharmacologically acceptable conventional carrier(s) or vehicle(s),specifically sterilized water, physiological saline, vegetable oils,emulsifying agents, suspending agents, and the like, to formpharmaceutical preparations, for example, solid, semi-solid or liquidpreparations (e.g., tablets, pills, troches, capsules., suppositories,cream, ointment, aerosol, powders, liquid preparations, emulsions,suspensions, and the like).

Administration to a patient may be achieved properly through nose, eye,external (local) site, rectum, lung (nose or oral injection), oral orparenteral (subcutaneous, intravenous or intramuscular) administration,or inhalation. Administration by injection may be achieved by aconventional method such as intra-arterial, intravenous, or subcutaneousinjection.

The dosage of the compound of the invention as a therapeutic agent maybe determined as a dose which shows a desired and satisfied therapeuticeffect. The therapeutically effective dose of the compound, for example,for oral administration, is usually in about 0.1-100 mg, preferably 1-16mg, a day. The effective single dose is chosen in the range of 0.001-1mg, preferably 0.01-0.16 mg, for 1 kg of the patient weight. Theabove-mentioned dose, however, may be altered depending on theindividual patient's body weight, age and condition, as well as methodfor administration. An appropriate dosage may be chosen properly by aperson with a usual experimental technique based on animal experimentaldata.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing showing that the platelet number in blood (n=5) isdecreased depending on the dosage of an HDAC inhibitor, Compound B.

FIG. 2 is a drawing showing that the megakaryocyte number in spleen(n=5) is increased depending on the dosage of an HDAC inhibitor,Compound B.

FIG. 3 is a drawing showing that the amount of expressed GATA-1 (n=5)for unit megakaryocytes in spleen in which GPIIb has been measured as aninternal standard is inhibited depending on the dosage of an HDACinhibitor, Compound B.

FIG. 4 shows a map of plasmid pGL3 IL-2 Pro43.

FIG. 5 shows a map of plasmid pGL3-IE Promoter.

FIG. 6 shows a map of plasmid pGL3-HSI-IE Promoter.

FIG. 7 is a drawing showing that the luciferase activity in IL-2reporter gene is inhibited depending on the concentration of Compound D.

FIG. 8 is a drawing showing that the cell growth in an IL-2 reportergene assay system does not depend on the concentration of Compound D.

FIG. 9 is a drawing showing that the luciferase activity in GATA-1reporter gene is inhibited depending on the concentration of Compound D.

FIG. 10 is a drawing showing that the cell growth in a GATA-1 reportergene assay system does not depend on the concentration of Compound D.

FIG. 11 shows a correlation between the immunosuppressive effect and thethrombocytopenia effect and between the IL-2 and GATA-1 transcriptioninhibitory effects by an HDAC inhibitor in rats.

FIG. 12 is a drawing showing a pattern of the influence on transcriptionof a GATA-1 gene by 2 kinds of HDAC inhibitors (2 concentrations,respectively).

FIG. 13 shows a pattern of the influence on transcription of 45 genesselected from 12,626 genes by 2 kinds of HDAC inhibitors (2concentrations, respectively), indicating that the transcriptioninhibitory pattern of these genes by HDAC inhibitors is similar to thatof GATA-1 gene.

FIG. 14(a) shows the binding sequence of C/EBPβ used in C/EBPβ reporterplasmid.

FIG. 14(b) shows the binding sequence of HSF1 (or HSF2) used in an HSEreporter plasmid.

FIG. 14(c) shows the binding sequence of STAT3 used in a pSTAT3 reporterplasmid.

FIG. 15 shows the sequence of IE promoter (mutant). The capital letterindicates the site into which mutation is introduced.

FIG. 16 shows the sequence of HSI (mutant) region. The capital letterindicates the site into which mutation is introduced.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention will be explained more specifically by Examples, which arenot intended as a limitation thereof. All of the prior technicalliteratures cited in the present specification are herein incorporatedby reference.

EXAMPLE 1 Confirmation of the Thrombocytopenia Effect Caused byAdministration of an HDAC Inhibitor

To male Lewis rats of 8 weeks of age was orally administered an HDACinhibitor Compound B for 7 days. Rats were divided into 3 groupsconsisting of a 0 dose group, 3.2 mg/kg dose group, and 10 mg/kg dosegroup, wherein one group comprised 5 rats. Three hours after the finaladministration, blood was collected and the number of platelets wascounted. FIG. 1 shows the number of platelets of each group thuscounted. It was recognized that the platelet number was reduceddepending on the dose of Compound B.

Subsequently, a sample of spleen was collected from the rats afterdrawing blood, and a part of the inferior margin was fixed with 4%paraformaldehyde (PFA) for counting the number of megakaryocytes toprepare a paraffin section in a conventional way, which section wasstained by HE (hematoxylin-eosin) staining. Each section was observedunder an object lens of 10 magnifications to count the number ofmegakaryocytes/section in a bright-field. The number was divided by anarea of the section to give the number of megakaryocytes in the spleen.FIG. 2 shows the average count of megakaryocytes in a group. It wasrecognized that the number of megakaryocytes in spleen was increaseddepending on the dose of Compound B.

EXAMPLE 2 Determination of GATA-1 mRNA in the Spleen of Rats to Which anHDAC Inhibitor was Administered

All the residual spleens after sampling in Example 1 were frozen andextracted with RNeasy (RNA extraction kit) to give total RNA astemplate, from which cDNA was synthesized by a Random Primer method.Subsequently, this cDNA was used as a template and the GATA-1 cDNA wasamplified by a real-time PCR (SYBR technique) using ABI Prism 7700. Theamount of GATA-1 mRNA was determined from its amplification curve.

In order to exactly determine the transcription amount of GATA-1 permegakaryocyte, it is necessary to use, as an internal standard for mRNAdetermination, a gene which is expressed specifically in megakaryocytesand of which the expression amount per megakaryocyte is not soinfluenced by administration of an HDAC inhibitor. Therefore, a mutationof gene expression influenced by an HDAC inhibitor in a cultured cell ofmegakaryocytic cell line, i.e., HEL cell, was analyzed using Gene Chip(Affymetrix), and glycoprotein IIb (GPIIb) was selected as an internalstandard.

FIG. 3 indicates the amount of expressed GATA-1 determined using GPIIbas an internal standard. Inhibition of GATA-1 transcription wasrecognized to depend on the dose of an HDAC inhibitor Compound B. Fromthe above experimental results, it was found that the inhibition ofGATA-1 transcription was observed in rats in which the platelet wasreduced by administration of an HDAC inhibitor.

EXAMPLE 3 Construction of a Reporter Gene Plasmid for IL-2 Reporter GeneAssay

A DNA fragment proximal to the transcription initiation point of humanIL-2 gene ranging from −674 to +54 (the main transcription initiationpoint of IL-2 gene is regarded as +1) was obtained by PCR using as atemplate a genomic DNA isolated from Jurkat cells of human T cellorigin. The sequences of the primers used in PCR are shown in SEQ IDNOS: 1 and 2 in Sequence Listing. These primers were designed based onthe IL-2 gene sequence (Locus code: HSIL05, Accession Number: X00695) asdescribed in GenBank gene database, in which at the end of primers arestriction enzyme recognition site was added in order to introduce itinto a vector for reporter gene assay. That is, the primers weredesigned so that a NheI recognition site was formed at the upstream sideof the promoter of the amplified DNA fragment and a HindIII recognitionsite was formed at the downstream side of the promoter. The DNA fragmentamplified by PCR was introduced into a cloning vector pCR4 (Invitrogen),and the base sequence of the insertion region was confirmed from theresulting plasmid. As a result, it was confirmed that the plasmid wasidentical to the promoter sequence of IL-2 gene as described in theabove GenBank, except for 3 sites of base substitution, 1 site ofinsertion of 2 bases, and 1 site of 1 base insertion. Total basesequence comprising 731 base pairs is shown in SEQ ID NO: 8. Thusresulting plasmid was cleaved at the Nhe I recognition site and HindIIIrecognition site, and the resulting fragment was inserted into the NheI-Hind III site of a vector pGL3 basic (Promega) for reporter gene assaycontaining a firefly luciferase gene. Thus, a plasmid pGL3 IL-2 Prowhich has at the upstream of firefly luciferase gene a DNA sequence of731 base pairs corresponding to the 728 base pair region proximal to thetranscription initiation point of human IL-2 gene ranging from −674 to+54 was obtained.

Subsequently, A DNA fragment proximal to the transcription initiationpoint of human IL-2 gene ranging from −378 to +54 was obtained by PCRusing pGL3 IL-2 Pro as a template. The sequences of the primers used inPCR are shown in SEQ ID NOS: 3 and 2 in Sequence Listing. The primer ofSEQ ID NO:3 were designed based on the DNA sequence introduced into theabove-mentioned pGL3 IL-2 Pro. At the end of the primers a restrictionenzyme recognition site was added in order to insert it into a vectorfor reporter gene assay. The primers are designed so that a NheIrecognition site is formed at the upstream side of the promoter of theamplified DNA fragment and a Hind III recognition site was formed at thedownstream side of the promoter. The DNA fragment amplified by PCR wasinserted into a cloning vector pCR4, and the base sequence of theinsertion region was confirmed using the resulting plasmid. Thisinsertion region was identical to the region ranging from −378 to +54 ofthe IL-2 promoter sequence as described in GenBank Locus code: HSIL05,except for 2 sites of base substitution and 1 site of insertion of 2bases. Total base sequence comprising 434 base pairs is shown in SEQ IDNO: 9. Thus resulting plasmid was cleaved at the Nhe I recognition siteand Hind III recognition site, and the resulting fragment was insertedinto the Nhe I-Hind III site of a vector pGL3 basic for reporter geneassay containing a firefly luciferase gene. Thus, a plasmid pGL3 IL-2Pro43 which had at the upstream of firefly luciferase gene a promotersequence of 434 base pairs corresponding to the 432 base pairs proximalto the transcription initiation point of human IL-2 gene ranging from−378 to +54 was obtained (FIG. 4).

EXAMPLE 4 Construction of a Reporter Gene Plasmid for GATA-1 ReporterGene Assay

A fragment of human GATA-1 gene of 819 base pairs proximal to thetranscription initiation point ranging from −789 to +30 was obtained byPCR using a human genome DNA as a template. In PCR, the primers asdescribed in SEQ ID NOS: 4 and 5 in Sequence Listing were used. Theseprimers were designed based on the GATA-1 gene sequence of AccessionNumber: AF196971 as described in GenBank gene database, in which at theend of primers a restriction enzyme recognition site was added in orderto insert it into a vector for reporter gene assay. Thus, a Bgl IIrecognition site was formed at the upstream side of the promoter of theamplified DNA fragment and a Hind III recognition site was formed at thedownstream side of the promoter. The DNA fragment amplified by PCR wasintroduced into a cloning vector pCR4. The base sequence of theinsertion region in the resulting plasmid was confirmed to be fullyidentical with the GATA-1 promoter sequence as described in GenBankAccession Number: AF196971. The total base sequence is shown in SEQ IDNO: 10. Thus resulting plasmid was cleaved at the Nhe I recognition siteand Hind III recognition site. The resulting fragment was inserted intothe Nhe I-Hind III site of a vector pGL3 basic for reporter gene assaycontaining a firefly luciferase gene. Thus, a plasmid pGL3-IE Promoterwhich had at the upstream of firefly luciferase gene a sequence of 819base pairs of GATA-1 gene promoter ranging from −789 to +30 was obtained(FIG. 5).

Subsequently, a fragment of 637 base pairs of human GATA-1 gene rangingfrom −3769 to −3133 at the upstream of transcription initiation pointwas prepared by PCR using a human chromosomal DNA as a template. In PCR,the primers as described in SEQ ID NO: 6 and 7 in Sequence Lisiting wereused. These primers were designed based on the GATA-1 gene sequence ofAccession Number: AF196971 as described in GenBank gene database, inwhich at the end of primers a restriction enzyme recognition site wasadded in order to insert it into a vector for reporter gene assay. Thus,a Kpn I recognition site was formed at the upstream side of the promoterof the amplified DNA fragment and a Nhe I recognition site was formed atthe downstream side of the promoter. The DNA fragment amplified by PCRwas inserted into a cloning vector pCR4, and the base sequence of theinsertion region in the resulting plasmid was confirmed to be fullyidentical with the sequence at the upstream of transcription initiationpoint of the GATA-1 gene as described in GenBank Accession Number:AF196971. The total base sequence consisting of 637 base pairs is shownin SEQ ID NO: 11. Thus resulting plasmid was cleaved at the Kpn Irecognition site and Nhe I recognition site, and the resulting DNAfragment was inserted into the Kpn I-Nhe I site of a vector pGL3 IEPromoter. Thus, a plasmid pGL3-HSI-IE Promoter which had at the upstreamof firefly luciferase gene a region of 637 base pairs ranging from −3769to −3133 and a sequence of 819 base pairs ranging from −789 to +30 atthe upstream of transcription initiation point of the GATA-1 gene wasobtained (FIG. 6).

EXAMPLE 5 Construction of a Screening System for an HDAC InhibitorHaving an IL-2 Transcription Inhibitory Effect

pGL3 IL-2 Pro43 (1 μg) obtained in Example 3 was mixed with 6 μg ofoptional carrier plasmid, and Jurkat cells (1×10⁷ cells) weretransformed therewith by electroporation (Voltage 300V; charge 975 μF;400 μL). After transformation, there was added 2.5 mL of 10%FBS-containing RPMI 1640 (10% FBS RPMI 1640), which was distributed to a96-well white plate at a rate of 50 μl/well. The plate was incubated at37° C. under 5% CO₂ in a condition of saturated humidity for 12 hours,and then 10% FBS RPMI 1640 medium containing Compound D (HDAC inhibitor)at a concentration of 4 times over the final concentration was added ata rate of 25 μL/well. Additionally, a mixture of phorbol 12-myristate13-acetate (PMA, SIGMA), ionomyicn (SIGMA) and anti-CD28 antibody(Pharmingen) in 10% FBS RPMI 1640 was added at a rate of 25 μL/well(final concentration, 50 ng/mL, 1 μg/mL and 75 ng/mL, respectively) tostimulate the Jurkat cells. After incubation at 37° C. under 5% CO₂ in acondition of saturated humidity for 12 hours, the luciferase activity inthe cells was measured with a multi-label counter (1420 MULTILABELCOUNTER ARVO SX, WALLAC) according to a manual of Bright-Glo™ LuciferaseAssay System (Promega Corporation). As a result, it was found thatluciferase was derived from pGL3 IL-2 Pro43 in response to stimulationof Jarkat cells with PMA, ionomycin and anti-CD28 antibody (FIG. 7).Further, it was found that the amount of expressed luciferase derivedtherefrom was inhibited dose-dependently by Compound D (FIG. 7). It wasconfirmed by Cell Counting Kit-8 (Dojindo Laboratories) that suppressionof the amount of expressed luciferase by Compound D was not a result ofsome damage of the cells by Compound D (FIG. 8). From theabove-mentioned results, it is confirmed that this assay system can beused in screening of an HDAC inhibitor having IL-2 transcriptioninhibitory effect.

EXAMPLE 6 Construction of a Screening System for an HDAC InhibitorCompound D Having GATA-1 Transcription Inhibitory Effect

Using 15 μg of pGL3-HSI-IE promoter obtained in Example 2, HEL cells(JCRB0062, Japanese Collection of Research Bioresources)(8.75×10⁶ cells)were transformed by electroporation (Voltage 1750V; charge 10 μF; 365μL). After transformation, there was added 2 mL of 10% FBS RPMI 1640,and the mixture was distributed to a 96-well white plate at a rate of 50μL/well. After incubation at 37° C. under 5% CO₂ in a condition ofsaturated humidity for 3 hours, 10% FBS RPMI 1640 medium containing anHDAC inhibitor Compound Data concentration of 2 times over the finalconcentration was added at a rate of 50 μL/well. After incubation at 37°C. under 5% CO₂ in a condition of saturated humidity for 8 hours, theluciferase activity in the cells was measured with a multi-label counteraccording to a manual of Bright-Glo™ Luciferase Assay System (PromegaCorporation). As a result, it was found that luciferase was derived frompGL3-HSI-IE promoter depending on a GATA-1 promoter, and further theamount of expressed luciferase was inhibited dose-dependently byCompound D (FIG. 9). It was confirmed by Cell Counting Kit-8 (DojindoLaboratories) that the inhibition of the amount of expressed luciferaseby Compound D was not a result of some damage of the cells by Compound D(FIG. 10). From the above-mentioned results, it was confirmed that thisassay system can be used in screening of an HDAC inhibitor having GATA-1transcription inhibitory effect.

EXAMPLE 7 Confirmation of the Rate of Platelet Decrease in the MinimumEffective Dose of an HDAC Inhibitor with an Immunosuppressive Effect inRat's Heart Transplantation

Nine HDAC inhibitory compounds selected at random as analytes (as shownin Tables 1 and 2) were evaluated in an allopatric heart-to-necktransplant model (Cuff method), wherein ACI rats were used as donors,and Lewis rats as recipients. Each compound was orally administered with10% HCO-60 (polyoxyl 60 hydrogenated castor oil)/water as solvent orsubcutaneously with 10% HCO-60/saline as solvent, once a daycontinuously for 14 days from the day of transplantation. Cardiac arrestwas judged as occurrence of rejection, and the number of days of takewas regarded as up to the day before the cardiac arrest. Since themedian of the number of take days is 5 days in a control group(solvent-administered group), the median over 8 days was judgedeffective, and the minimum effective dose was determined, respectively.

Next, the analyte was administered to normal Lewis rats at the minimumeffective dose respectively determined above in the same administrationroute as in a pharmacological test once a day, totally 7 times. The dayafter the final administration, blood was collected, and the number ofplatelet was counted in individuals. Thus, the rate (%) of decrease tothe control group was calculated from the mean value in each group(n=4).

Tables 1 and 2 indicate the minimum effective dose and the rate ofplatelet decrease thus obtained in a rat's heart transplant model with avariety of HDAC inhibitors. The dose judged as the minimum effectivedose is indicated by an underline and boldface. TABLE 1 Effect in Rat'sheart Rate of transplantation platelet Com- Admn. Dose Survival Mediandecrease pound Route (mg/kg) days (Days) (%) Control p.o. 5, 5, 5, 5, 5,5, 6, 6 5  0 (solvent) Comp. A s.c. 10 5, 5, 5, 5, 6, 6, 10 5 32 17, 17,18, 19, 19, 18.5 75 21 Comp. B p.o. 1 5, 5, 5, 6, 6, 6, 9 6 3.2 7, 7,11, 11, 11, 11, 11 37 27 Comp. C p.o. 3.2 4, 4, 4, 5, 5, 5, 5, 5, 5 6 105, 5, 5, 5, 12, 15, 8.5 44 17, 20 Comp. D p.o. 3.2 5, 5, 6, 6, 6, 6, 6 610 18, 19, 19, 1920, 19 40 21, 24

TABLE 2 Effect in Rat's heart Rate of transplantation platelet Com-Admn. Dose Survival Median decrease pound Route (mg/kg) days (Days) (%)Comp. E p.o. 0.32 5, 5, 5, 5, 5, 5, 5, 5, 5 9 1 5, 6, 7, 11, 17, 18, 1134 20 Comp. G s.c. 0.56 5, 5, 6, 9 5.5 1.8 5, 9, 9, 9 9 23 Comp. F s.c.0.32 4, 5, 10, 10 7.5 1 21, 21, 23, 23 22 27 Comp. H s.c. 0.32 5, 6, 6,6 6 1 12, 15, 16, 17, 20, 17 8.5 21, 22 Comp. I s.c. 0.56 N.T. 1.8 18,18, 19, 19 18.5 17

EXAMPLE 8 Method for Selecting an HDAC Inhibitor Which has anImmunosuppressive Effect with a Less Thrombocytopenia Effect

Compounds as shown in Table 3 were used as analytes for assay using amethod for screening an HDAC inhibitor having an IL-2 transcriptioninhibitory effect as shown in Example 3. The concentration of a druginhibiting by 50% of the expression of luciferase was calculated as theIC50 value (IL-2 IC50) for the inhibition of IL-2 transcription from adose-response curve when the amount of expression of luciferase in theabsence of an analyte was regarded as 100% (Table 3). Subsequently, thecompounds as shown in Table 1 were used as analytes for assay using amethod for screening an HDAC inhibitor having an GATA-1 transcriptioninhibitory effect as shown in Example 4. The concentration of a druginhibiting by 50% of the expression of luciferase was calculated as theIC50 value (GATA-1 IC50) for the inhibition of GATA-1 transcription froma dose-response curve when the amount of expression of luciferase in theabsence of an analyte was regarded as 100% (Table 3). Further, the G/Iratio was calculated by dividing GATA-1 IC50 by IL-2 IC50 (Table 3). Therate of platelet decrease at a minimum effective dose of an analyte inthe rat's heart transplantation obtained in Example 5 and the G/I ratioobtained in this example were plotted (FIG. 11) to give a goodcorrelation. In addition, it was found that the compounds showing a G/Iratio of 5 or more reduced the rate of platelet decrease to 30% or lowerat a minimum effective dose at which the compounds showed animmunosuppressive effect in a rat's heart transplant model. From theabove results, it was confirmed that this assay system is suitable forscreening an HDAC inhibitor having an immune effect with a lessthrombocytopenia effect. TABLE 3 Reporter Gene Assay IC50 (nM) RatioHDAC Inhibitor IL-2 GATA-1 GATA-1/IL-2 Compound A 342.9 475.7 1.39Compound B 32.3 117.3 3.63 Compound C 8.7 32.6 3.73 Compound D 27.5117.3 4.27 Compound E 30.7 150.5 4.90 Compound F 23.1 128.0 5.53Compound G 14.8 91.0 6.16 Compound H 6.1 37.8 6.20 Compound I 19.6 148.67.58

EXAMPLE 9 Search of a Transcriptional Factor Relating to aThrombocytopenia Effect by an HDAC Inhibitor Using a GeneChip Analysis

From the genes expressed in megakaryocytes, a gene of which theexpression is inhibited by an HDAC inhibitor in the same pattern asGATA-1 was screened and grouped with a GeneChip (Affymetrix) to search atranscriptional factor common to these gene groups.

Human megakaryocytic culture cells, HEL cells, were treated with 2 kindsof HDAC inhibitors showing a thrombocytopenia effect (500 nM and 1500 nMof Compound A, and 100 nM and 300 nM of Compound D) for 8 hours, andthen extracted with RNeasy Kit (QIAGEN) to give the total RNA. The totalRNA was used as a template to synthesize cDNA using a Superscript ChoiceSystem (Invitrogen) and further synthesize a fluorescence labeled cRNAusing BioArray High Yield RNA transcript Labeling Kit (EnzoDiagnostics). The resulting fluorescence labeled cRNA was used as aprobe to investigate the expression pattern of 12,626 genes using aHuman Genome U95A Array (Affymetrix).

The effect of the above 2 HDAC inhibitors influencing the transcriptionof GATA-1 gene showed a pattern as shown in FIG. 12. This expressionpattern was comparatively analyzed by a gene expression analyzing tool,GeneSpring (Silicon Genetics). As a result, 45 types of genes was found,of which the transcription was inhibited by the HDAC inhibitors in thesame pattern as the GATA-1 gene (FIG. 13). From these genes, further,candidate genes as transcriptional factor expected to have a functionassociated with differentiation of blood corpuscles or candidate genesexpected as megakaryocyte marker genes were selected, and the following10 genes were obtained.

-   SCL (GenBank Accession Number M63589: P. D. Aplan et al., (1990)    Mol. Cell. Biol. 10(12), 6426-6435)-   NF-E2 (GenBank Accession Number S77763: C. Pischedda et al., (1995)    Proc. Natl. Acad. Sci. U.S.A. 92(8), 3511-3515)-   EKLF (GenBank Accession Number U65404: J. H. van Ree et al., (1997)    Genomics 39(3), 393-395)-   Pleckstrin (GenBank Accession Number X07743: M. Tyers et al., (1988)    Nature 333 (6172), 470-473)-   Thrombin-R (GenBank Accession Number M62424: T. K. Vu et al., (1991)    Cell 64(6), 1057-1068)-   LMO2 (GenBank Accession Number X61118: B. Royer-Pokora et    al., (1991) Oncogene 6(10), 1887-1893)-   PU.1 (GenBank Accession Number X52056: D. Ray et al., (1990)    Oncogene 5(5), 663-668)-   Fli-1 (GenBank Accession Number M98833: D. K. Watson et al., (1992)    Cell Growth Differ. 3(10), 705-713)-   AML1 (GenBank Accession Number D43968: H. Miyoshi et al., (1991)    Proc. Natl. Acad. Sci. U.S.A. 88(23), 10431-10434)-   TCF11 (GenBank Accession Number L24123: J. Y. Chan et al., (1993)    Proc. Natl. Acad. Sci. U.S.A. 90(23), 11371-11375)

Subsequently, the sequence information on the 2 kb upstream oftranscription initiation sites of the above-mentioned respective geneswas downloaded from the human genome sequence information (GenomeAnnotation, NCBI Locus Link), and the database of transcriptionalfactors (The Transcription Factor Database, TRANSFAC® Professional 6.3,BIOBASE Biological Databases/Biologische Datenbanken GmbH) was searchedbased on a prediction assisting system for transcriptional controlregions TRANSFAC (E. Wingender et al., (2000) Nucleic Acids Research 28,316-319, TRANSFAC: an integrated system for gene expression regulation)to investigate the transcriptional factor binding sequences commonlypresent in the respective promoter sequences of human origin. In thisprocedure, in the transcriptional control region within 2 kb upstream ofthe transcription initiation point of the above-mentioned respectivegenes of which the expression was inhibited by an HDAC inhibitor, thetranscriptional factor binding sequences commonly present in almost ofgenes, more particularly, the sequences commonly present in either of IEpromoter and HSI region in GATA-1 gene, were mainly selected. As aresult, the following 4 sequences (1) to (4) were found as candidates ofthe transcriptional factors involved in the inhibition of expression ofthe above-described genes by an HDAC inhibitor.

(1) STAT3: signal transducer and activator of transcription 3(acute-phase response factor)

(2) C/EBPβ: CCAAT/enhancer binding protein (C/EBP), beta

(3) HSF1 (or HSF2): heat shock transcription factor 1 (or heat shocktranscription factor 2)

(4) GATA-1: GATA binding protein 1

EXAMPLE 10 Construction of a Reporter Gene (Luciferase) Vector Having aResponsive Sequence Recognized by STAT3, C/EBPβ and HSF1

Among the 4 candidates of transcriptional factors, STAT3, C/EBPβ, HSF1and GATA-1, involved in the transcriptional control of the GATA-1 geneby an HDAC inhibitor, for the 3 transcriptional factors, STAT3, C/EBPβand HSF1 (or HSF2), a reporter gene (luciferase) vector having aresponsive sequence recognized by the respective transcriptional factorswas constructed.

(C/EBPβ Reporter Plasmid)

CEBP-11 and CEBP-12 represented by the following sequences weredesigned, in which the so far reported 5 consensus sequences recognizedby C/EBPβ (M. Rosati et al., (2001) The Journal of Immunology 167,1654-1662, CCAAT-Enhancer-Binding Protein β (C/EBPβ) Activates CCR5Promoter: Increased C/EBPβ and CCR5 in T Lymphocytes from HIV-1-InfectedIndividuals) (S. Osada et al., (1996) The Journal of BiologicalChemistry 271, 3891-3896, DNA Binding Specificity of the CCAAT/EnhancerBinding Protein Transcription Factor Family) were connected in series togive synthetic DNAs (Amersham Biosciences). CEBP-11:5′-CGCGTTGAGCAAGACTTGAGCAAGTACTTGAGCAAGCGTTGAGCAAG GCTTGAGCAAGC-3′CEBP-12: 5′-TCGAGCTTGCTCAAGCCTTGCTCAACGCTTGCTCAAGTACTTGCTCAAGTCTTGCTCAA-3′

(HSE Reporter Plasmid)

According to the HSE (heat shock promoter element) used in pHSE-TAL-Luc(BD Biosciences Clontech), HSE-11 and HSE-12 as shown by the followingsequences were designed to give synthetic DNAs (Amersham Biosciences).HSE-11: 5′- CGCGTCTAGAATGTTCTAGATCTAGAACATTCTAGCTAGAATGTTCTA GAC-3′HSE-12: 5′- TCGAGTCTAGAACATTCTAGCTAGAATGTTCTAGATCTAGAACATTCT AGA-3′

The above-described 2 synthetic DNAs were mixed and annealed to give afragment of HSE sequence as shown in FIG. 14(b). This fragment wasligated to a fragment of about 4.9 kb prepared from pTA-Luc (BDBiosciences Clontech) by treatment with MluI/XhoI to give pHSE-TA-Luc.On the other hand, pTAL-Luc (BD Biosciences Clontech) was treated withBglII/SphI to give a fragment of about 0.7 kb coding for a TAL promotersequence and firefly luciferase gene N-terminal region. This fragmentwas ligated to a fragment of about 4.2 kb prepared by treatment ofpHSE-TA-Luc with BglII/SphI to give a HSF1 reporter plasmid pHSE-TAL-Lucwhich contains a TAL promoter sequence and firefly luciferase gene atthe downstream of the HSE sequence.

(pSTAT3 Reporter Plasmid)

pTAL-Luc (BD Biosciences Clontech) was treated with BglII/SphI to give afragment of about 0.7 kb coding for TAL promoter sequence and fireflyluciferase gene N-terminal region. This fragment was ligated to afragment of about 4.2 kb prepared from pSTAT3-TA-Luc (BD BiosciencesClontech) by treatment with BglII/SphI to give a STAT3 reporter plasmidpSTAT3-TAL-Luc which contains a TAL promoter sequence and fireflyluciferase gene at the downstream of the STAT3 binding sequence as shownin FIG. 14(c).

EXAMPLE 11 Effect of an HDAC Inhibitor on the Transcriptional Activityof STAT3, C/EBPβ and HSF1

HEL cells (8.75×10⁶ cells) were transformed with 15 μg each ofpC/EBPβ-TAL-LUc, pHSE-TAL-Luc, and pSTAT3-TA-Luc prepared in Example 10by electroporation (voltage 1750V; Charge 10 μF, 365 μL). Aftertransformation, the transformants respectively were suspended in 2 mL of10% FBS RPMI 1640, and distributed into a 96-well white plate at a rateof 50 μL/well. The plate was incubated at 37° C. under 5% CO₂ in acondition of saturated humidity for 3 hours, and then 10% FBS RPMI 1640medium containing Compound D (HDAC inhibitor) at a concentration of 2times over the final concentration was added at a rate of 50 μL/well.After incubation at 37° C. under 5% CO₂ in a condition of saturatedhumidity for 8 hours, the luciferase activity in the cells was measuredwith a multi-label counter according to a manual of Bright-Glo™Luciferase Assay System (Promega Corporation). As a result, it was foundthat luciferase is derived from pC/EBPβ-TAL-LUc, pHSE-TAL-Luc, andpSTAT3-TA-Luc depending on the transcriptional activity of C/EBPβ, HSF1and STAT3, respectively. Their induction of luciferase activity was notinhibited by Compound D (Table 4). From the above results, it waselucidated that the transcriptional inhibitory effect of GATA-1 by anHDAC inhibitor is not due to the transcriptional inhibitory effect ofC/EBPβ, HSF1 (or HSF2) and STAT3. TABLE 4 Luciferase activity (%)Compound D pC/EBPβ- (nM) TAL-LUc pHSE-TAL-Luc pSTAT3-TAL-Luc 0 100 100100 3 173 ± 3 163 ± 7 166 ± 10 10 277 ± 4 258 ± 9 238 ± 7  30 367 ± 9 338 ± 18 316 ± 16 100 388 ± 5  355 ± 12 334 ± 18 300 379 ± 1 361 ± 3326 ± 13Relative activity wherein the average luciferase activity is 100 with noaddition of Compound DMean ± Standard error (n = 3)

EXAMPLE 12 Construction of a Reporter Gene (Luciferase) Vector Having aMutant Promoter in Which Mutation is Introduced into all of the GATA-1Recognition Sites in the GATA-1 Promoter

GATA-1 promoter has therein a responsive sequence (GATA sequence)recognized by GATA-1 per se. In order to elucidate whether thetranscriptional control of the GATA-1 gene by an HDAC inhibitor isassociated with the transcriptional control system through the GATAsequence, a promoter in which the responsive sequence recognized byGATA-1 per se was eliminated by introducing a mutation into the GATAsequence was artificially constructed and ligated to a reporter gene togive a vector.

That is, an IE promoter (mutant) of about 0.84 kb in which a mutationwas introduced at the 5 sites of the GATA sequence in the IE promoterregion of GATA-1 gene was artificially constructed by means of PCR. Inthis procedure, pGL3-HSI-IE DNA prepared in Example 4 was used as atemplate in the first PCR, and using synthetic primers corresponding tothe both ends of the promoter region and the respective sequences havingthe mutation, fragments to be placed between the terminals of the regionor between the respective sites having the mutation were amplified.Thus, the resulting PCR products were mixed and used as templates in thesubsequent PCR reaction to select appropriate members as syntheticprimers and amplify their longer region. By repeating this PCR, a PCRfragment corresponding to the total IE promoter region was obtainedusing the synthetic primers corresponding to both terminals of thepromoter region. This fragment was inserted into pCR4-TOPO (Invitrogen)to determine the base sequence. Thus, the mutation was confirmed to beintroduced as designed. FIG. 15 shows the sequence of the IE promoter(mutant) thus confirmed. The portion of the mutation is indicated byunderlines, and both terminals have the BglII site and HindIII site asintroduced. The BglII-HindIII fragment having the total IE promoter(mutant) was inserted between the BglII site and the HindIII site ofpGL3 to give pGL3-IE promoter (mutant).

By the same PCR technique, the HSI (mutant) region of about 0.65 kb inwhich a mutation was introduced at the 2 sites of the GATA sequence ofthe HSI region in GATA-1 gene was artificially constructed usingpGL3-HSI-IE DNA prepared in Example 4 as a template in the first PCR.The resulting PCR fragment was inserted into pCR4-TOPO (Invitrogen) todetermine the base sequence and confirm the mutation introduced. FIG. 16shows the sequence of the HSI (mutant) region thus confirmed. Theportion of the mutation is indicated by underlines, and both terminalsrespectively have the MluI site and BglII site as introduced. TheMluI-BglII fragment having the total HSI (mutant) region was insertedbetween the MluI site and the BglII site of pTAL-Luc to givePTAL-HSI(mutant)-Luc. In addition, the KpnI-BglII fragment having thetotal HSI (mutant) region of pTAL-HSI(mutant)-Luc was inserted betweenKpnI-BglII of pGL3-IE promoter (mutant) to give pGL3-HSI-IE Promoter(mutant) having the total IE promoter (mutant) and the total HSI(mutant) region.

EXAMPLE 13 Effect of an HDAC Inhibitor on the GATA-1 TranscriptionalActivity

Using 15 μg each of pGL3-HSI-IE Promoter prepared in Example 4 andpGL3-HSI-IE Promoter (mutant) prepared in Example 12, HEL cells(8.75×10⁶ cells) were transformed by electroporation (Voltage 1,750V,Charge 10 μF, 365 μL). After transformation, the transformantsrespectively were suspended in 2 mL of 10% FBS RPMI 1640, anddistributed to a 96-well white plate at a rate of 50 μL/well. The platewas incubated at 37° C. under 5% CO₂ in a condition of saturatedhumidity for 3 hours, and then 10% FBS RPMI 1640 medium containing anHDAC inhibitor, i.e., Compounds A, C or D at a concentration of 2 timesover the final concentration was added at a rate of 50 μL/well. Afterincubation at 37° C. under 5% CO₂ in a condition of saturated humidityfor 8 hours, the luciferase activity in the cells was measured with amulti-label counter according to a manual of Bright-Glo™ LuciferaseAssay System (Promega Corporation). As a result, it was found thatluciferase was derived from pGL3-HSI-IE Promoter and pGL3-HSI-IEPromoter (mutant), respectively, depending on the GATA-1 promoter, butthe activity derived from the mutant promoter was lower than that of thewild-type promoter. In addition, the luciferase activity derived fromthe mutant promoter was not inhibited by Compounds A, C and D differingfrom the wild-type (Table 5).

The above results indicate that the HDAC inhibitors, Compounds A, C andD, inhibit the function of transcriptional control system involved inthe GATA sequence on the GATA-1 promoter to inhibit the function oftranscriptional activation. The GATA sequence is known to be recognizedby GATA-1 itself; this fact suggests that Compounds A, C and D mightinhibit the function of the transcriptional activation of GATA-1 factoritself. TABLE 5 Relative luciferase activity (RLU) Wild type GATA-1Mutant GATA-1 promoter promoter Compound A (+) 211 ± 13 301 ± 30 (−) 586± 44 317 ± 7  Compound C (+) 218 ± 5  311 ± 8  (−) 609 ± 43 326 ± 11Compound D (+) 203 ± 17 290 ± 23 (−) 681 ± 68 335 ± 10Mean ± Standard error (n = 3)

INDUSTRIAL APPLICABILITY

According to the method of the invention, it is possible to rapidlyscreen a compound having a strong IL-2 transcription inhibitory activitysimultaneously with a weak GATA-1 transcription inhibitory activity, byusing reporter assay systems using 2 species of cells. It is alsopossible to rapidly screen a compound having a weak GATA-1 transcriptioninhibitory activity from HDAC inhibitors. The compounds selected by sucha method are expected to be candidates for creating highly safe drugswith less thrombocytopenia effect in much higher probability thanbefore. Thus, the method of the invention is very useful in studies forcreation of new drugs.

1. A method for selecting an immunosuppressive agent with a lessthrombocytopenia effect, said method comprising the following items (1)and (2): (1) selecting compounds having an immunosuppressive activity;and (2) selecting a compound having a weak GATA-1 transcriptioninhibitory activity from the compounds selected in (1).
 2. A method forselecting an immunosuppressive agent with a less thrombocytopeniaeffect, said method comprising the following items (1) to (3): (1)measuring an immunosuppressive activity of an analyte; (2) measuring aGATA-1 transcription inhibitory activity of the analyte; and (3)comparing the immunosuppressive activity determined in (1) with theGATA-1 transcription inhibitory activity determined in (2) to select animmunosuppressive agent with a less thrombocytopenia effect.
 3. A methodfor selecting an immunosuppressive agent with a less thrombocytopeniaeffect, said method comprising the following items (1) to (3): (1)measuring an IL-2 transcription inhibitory activity in a test cell inthe coexistence of the test cell and an analyte; (2) measuring an GATA-1transcription inhibitory activity in a test cell in the coexistence ofthe test cell and an analyte; and (3) comparing the IL-2 transcriptioninhibitory activity determined in (1) with the GATA-1 transcriptioninhibitory activity determined in (2) to select an immunosuppressiveagent with a less thrombocytopenia effect.
 4. A method for selecting animmunosuppressive agent with a less thrombocytopenia effect, said methodcomprising the following items (1) to (3): (1) measuring an IL-2transcription inhibitory activity in a test cell into which an IL-2reporter gene has been introduced in the coexistence of the test celland an analyte; (2) measuring a GATA-1 transcription inhibitory activityin a test cell into which a GATA-1 reporter gene has been introduced inthe coexistence of the test cell and an analyte; and (3) comparing theIL-2 transcription inhibitory activity determined in (1) with the GATA-1transcription inhibitory activity determined in (2) to select animmunosuppressive agent with a less thrombocytopenia effect.
 5. A methodfor selecting an immunosuppressive agent with a less thrombocytopeniaeffect as claimed in claim 4, comprising measuring a (IL-2 IC50) valueas an IL-2 transcription inhibitory activity, measuring a (GATA-1 IC50)value as a GATA-1 transcription inhibitory activity, and comparing boththe values.
 6. A method for selecting an immunosuppressive agent with aless thrombocytopenia effect as claimed in claim 5, comprising selectinga compound having the (GATA-1 IC50)/(IL-2 IC50) value of 5 or more.
 7. Amethod as claimed in any of claims 4 to 6, wherein the GATA-1 reportergene comprises the transcriptional control region of human GATA-1 geneand a reporter gene.
 8. A method as claimed in any of claims 4 to 7,wherein the GATA-1 reporter gene comprises a sequence of the region from−3769 to −3133 upstream of the transcription initiation point andsequence of the region from −789 to +30 proximal to the transcriptioninitiation point of human GATA-1 gene.
 9. A method as claimed in any ofclaims 4 to 8, wherein the IL-2 reporter gene comprises thetranscriptional control region of IL-2 gene and a reporter gene.
 10. Amethod as claimed in any of claims 4 to 9, wherein the IL-2 reportergene comprises a sequence of the region from −378 to +54 proximal to thetranscription initiation point of human IL-2 gene.
 11. A method asclaimed in any of claims 4 to 10, wherein the test cell into which aGATA-1 reporter gene is introduced is a human megakaryocytic cellstrain.
 12. A method as claimed in any of claims 4 to 11, wherein thetest cell into which an IL-2 reporter gene is introduced is a human Tcell-derived cell strain stimulated by phorbol 12-myristate 13-acetate,ionomycin and anti-CD28 antibody, and the test cell into which a GATA-1reporter gene is introduced is a human megakaryocytic cell strain.
 13. Amethod as claimed in claim 12, wherein the human T cell-derived cellstrain is a Jurkat cell.
 14. A method as claimed in claim 11, whereinthe human megakaryocytic cell strain is a HEL cell.
 15. A method asclaimed in claim 12, wherein the human megakaryocytic cell strain is aHEL cell.
 16. A method as claimed in any of claims 4 to 15, wherein thereporter gene is a firefly luciferase gene.
 17. A method for selectionas claimed in any of claims 1 to 16, wherein the analyte is an HDACinhibitor.
 18. A method for selecting an immunosuppressive agent with aless thrombocytopenia effect, comprising measuring the amount ofexpression of GATA-1 protein.
 19. A method for selecting animmunosuppressive agent with a less thrombocytopenia effect, said methodcomprising the following items (1) to (3): (1) measuring the amount ofexpression of IL-2 protein; (2) measuring the amount of expression ofGATA-1 protein; and (3) comparing both the amounts of expression toselect an immunosuppressive agent with a less thrombocytopenia effect.20. A kit for measurement in selecting an immunosuppressive agent with aless thrombocytopenia effect, comprising the following items (1) and(2): (1) a DNA construct containing a GATA-1 reporter gene; and (2) atest cell of megakaryocytic cell line.
 21. A kit for measurement inselecting an immunosuppressive agent with a less thrombocytopeniaeffect, comprising the following items (1) to (4): (1) a DNA constructcontaining an IL-2 reporter gene; (2) a DNA construct containing aGATA-1 reporter gene; (3) a test cell of T-cell line; and (4) a testcell of megakaryocytic cell line.
 22. An HDAC inhibitor with a lessthrombocytopenia effect, said inhibitor being selected by a method asclaimed in claim
 17. 23. An immunosuppressive agent with a lessthrombocytopenia effect, said agent being selected by a method forselection as claimed in any of claims 1 to
 16. 24. An immunosuppressiveagent for treatment of inflammatory disorders, diabetes mellitus,diabetic complications, homozygous thalassemia, fibrosis, cirrhosis,acute promyelocytic leukemia, protozoal infections, organ transplantrejection, autoimmune diseases, and tumors, said agent comprising as anactive ingredient an HDAC inhibitor as claimed in claim
 22. 25. Atherapeutic agent for treatment of inflammatory disorders, diabetesmellitus, diabetic complications, homozygous thalassemia, fibrosis,cirrhosis, acute promyelocytic leukemia, protozoal infections, organtransplant rejection, autoimmune diseases, and tumors, said agentcomprising as an active ingredient an immunosuppressive agent as claimedin claim
 23. 26. A therapeutic method for treatment of inflammatorydisorders, diabetes mellitus, diabetic complications, homozygousthalassemia, fibrosis, cirrhosis, acute promyelocytic leukemia,protozoal infections, organ transplant rejection, autoimmune diseases,and tumors, said method comprising administering an immunosuppressiveagent with a less thrombocytopenia effect containing as an activeingredient an HDAC inhibitor as claimed in claim
 22. 27. A therapeuticmethod for treatment of inflammatory disorders, diabetes mellitus,diabetic complications, homozygous thalassemia, fibrosis, cirrhosis,acute promyelocytic leukemia, protozoal infections, organ transplantrejection, autoimmune diseases, and tumors, said method comprisingadministering an immunosuppressive agent with a less thrombocytopeniaeffect containing as an active ingredient an immunosuppressive agent asclaimed in claim
 23. 28. Use of an HDAC inhibitor as claimed in claim 22in manufacture of a therapeutic agent for treatment of inflammatorydisorders, diabetes mellitus, diabetic complications, homozygousthalassemia, fibrosis, cirrhosis, acute promyelocytic leukemia,protozoal infections, organ transplant rejection, autoimmune diseases,and tumors.
 29. Use of an immunosuppressive agent as claimed in claim 23in manufacture of a therapeutic agent for treatment of inflammatorydisorders, diabetes mellitus, diabetic complications, homozygousthalassemia, fibrosis, cirrhosis, acute promyelocytic leukemia,protozoal infections, organ transplant rejection, autoimmune diseases,and tumors.